Human pluripotent hematopoietic colony stimulating factor

ABSTRACT

Highly purified Pluripotent hematopoietic colony-stimulating factor (pluripotent CSF), a glycoprotein (MW 19,600) constitutively produced by human tumor cells has been highly purified from low serum-containing conditioned medium to apparant homogeneity. Pluripotent CSF supports the growth of human mixed colonies (CFU-GEMM), granulocyte-macrophage colonies (CFU-GM), early erythroid colonies (BFU-E) and induces differentiation of human leukemic cells. The specific activity of the purified pluripotent CSF in the CFU-GM assay is 1.5×10 8  U/mg protein.

This work was done in part with government funding under United StatesPublic Health Service Grants CA-32516, HL-31780, CA-20194, CA-23766 andCA-00966. Therefore the government has certain rights in this invention.

This is a continuation of application Ser. No. 08/132,240, filed on Oct.6, 1993, now abandoned, which was a continuation of application Ser. No.06/835,270 filed Mar. 7, 1986, now abandoned, which was acontinuation-in-part application of co-pending application Ser. No.716,844 filed Mar. 28, 1985 now abandoned.

BACKGROUND

This application concerns human pluripotent colony stimulating factor(P-CSF) also known as pluripoietin.

Abbreviations

CFU-GEMM: Colony forming unit--granulocyte, erythroid, macrophage,megakaryocyte.

CFU-GM: Colony forming unit--granulocyte-macrophage BFU-E: erythroidburst forming unit

GM-CSF: Granulocyte-macrophage colony stimulating factor

Colony-stimulating factors (CSFs) are hormone-like glycoproteinsproduced by a variety of tissues and tumor cell lines which regulatehematopoiesis and are required for the clonal growth and maturation ofnormal bone marrow cell precursors in vitro (Burgess, A. W., et al.(1980) Blood 56:947-958; Nicola, N. A., et al. (1984) Immunology Today5:76-81). In contrast to the murine system (Nicola, N. A., et al. (1983)J. Biol. Chem. 258:9017-9021; Ihle, J. N., et al. (1982) J. Immunol.129:2431-2436; Fung, M. C., et al. (1984) Nature 307:233-237; Gough, N.M., et al. (1984) Nature 309:763-767), human CSFs have been less wellcharacterized, both biologically and biochemically (Nicola, N. A., etal. (1979) Blood 54:614-627; Wu, M. C., et al. (1980) J. Clin. Invest.65:772-775; Golde, D. W., et al. (1980) Proc. Nat'l. acad. Sci. USA77:593-596; Lusis, A. J., et al. (1981) Blood 57:13-21; Abboud, C. N.,et al. (1981) Blood 58:1148-1154; Okabe, T., et al. (1982) J. Cell.Phys. 110:43-49). Purification to apparent homogeneity has only beenreported for macrophage active CSF (CSF- 1) (Das, S. K., et al. (1981)Blood 58:630-641; Das, S. K., et al. (1982) J. Biol. Chem.257:13679-13684) and erythroid potentiating activity [Westbrook, C. A.et al. J. Biol. Chem. 259:9992-9996 (1984)] and forgranulocyte-macrophage CSF (GM-CSF) [Gasson, J. C., et al. Science226:1339-1342 (1984)], but not for human pluripotent CSF.

Assays are available to detect human clonogenic precursors that giverise to cells of the erythroid, granulocytic, megakaryocytic, macrophage(CFU-GEMM) (Fauser, A. A., et al. (1978) Blood 52:1243-1248; Fauser, A.A., et al. (1979) Blood 53:1023-1027) and possibly lymphoid (Messner, H.A., et al. (1981) Blood 58:402-405) lineages. CSFs with activities onthese multipotential progenitor cells (pluripotent CSF or P-CSF) areproduced by mitogen- or antigen activated T lymphocytes (Ruppert, S., etal. (1983) Exp. Hematol. 11:154-161) and constitutively by human tumorcell lines such as the SK-HEP-1 human hepatoma cell line (J. Gabrilove,K. Welte, Li Lu, H. Castro-Malaspina, M. A. S. Moore, Blood, in Pressand hereby incorporated by reference); the 5637 bladder carcinoma cellline (reported herein and in Proc. Nat'l. Acad. Sci. 82:1526-1530 1985hereby incorporated by reference); and by the HTLV-transformed lymphoidcells (Fauser, A. A., et al. (1981) Stem Cells 1:73-80; Salahuddin, S.Z., et al. (1984) Science 223:703-707). Pluripotent CSF is involved inthe proliferation and differentiation of pluripotent progenitor cellsleading to the production of all major blood cell types. This istherefore a broad spectrum CSF. It also induces differentiation ofleukemic cells.

SUMMARY

This application concerns human puripotent colony stimulating factor CSFfor the stimulation of proliferation and differentiation of pluripotentprogenitor cells to all major blood cell types which is purified toapparent homogeneity. Its biological effects include the induction offunctional markers of differentiation of normal and leukemic cells.

DESCRIPTION OF THE DRAWINGS

Figure one shows ion-exchange chromatography of 5637 conditioned medium(CM) followed by the Gel filtration chromatograph shown in figure two.

Figure three shows pooled gel filtration eluants on HPLC (reversephase). FIG. 4 shows SDS-PAGE whereas FIG. 5 shows preparative SDS-PAGEand FIG. 6 isoelectrofocusing of the purified pluripotent CSF.

DESCRIPTION DESCRIPTION OF THE DRAWINGS

FIG. 1: Ion exchange chromatography

One liter dialyzed ammonium sulfate-precipitate of 5637 CM was appliedin 0.05M Tris/Hcl, pH 7.8, on a 1 L DEAE cellulose (DE 52) column. Boundproteins were eluted with a linear gradient of NaCl (0.05-0.3M) in 0.05MTris/Hcl, pH 7.8, as indicated (-). The elution of proteins wasmonitored by absorption at 280 nm (.-.) and each fraction was tested forCSF activities (GM-CSF activity: Δ). Proteins from the first peak ofGM-CSF activity eluted from the column gave rise to mixed colonies in aCFU-GEMM assay and were used for further purification (pluripotent CSF).

FIG. 2: Gel filtration chromatography

The pluripotent CSF containing concentrated pool of DEAE cellulosechromatography was loaded on an AcA 54 Ultrogel column (2.6×90 cm) andeluted with PBS. Arrows denote the elution points of bovine serumalbumin (MW 68,000), and chymotrypsinogen (MW 25,000). The elution ofproteins was monitored by absorption at 280 nm (.-.) and each fractionwas tested for pluripotent CSF activity (GM-CSF activity: Δ).

FIG. 3: Reverse phase high-performance liquid chromatography (HPLC)

The pooled fractions with pluripotent CSF activities eluted from the gelfiltration column were acidified to pH 4.0 and loaded onto a C 18(uBondapak, Waters) column. The bound proteins were eluted with a lineargradient of 1-propanol in 0.9M acetic acid/0.2M pyridine, pH 4.0. Theelution of proteins were monitored by absorption at 280 nm (-) and eachfraction was tested for pluripotent CSF activity (GM-CSF activity: Δ).

FIG. 4: SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

The pluripotent CSF eluted from the HPLC column (200 ng; peak fraction)was lyophilized and treated with 1% SDS in 0.0625M Tris/HCl, pH 6.8, and20% glycerol, under reducing conditions (5% 2-mercaptoethanol) for onehour at 37° C. and then applied to a 15% polyacrylamide gel. Afterelectrophoresis, the protein bands were visualized by the silverstaining technique.

FIG. 5: Preparative SDS-PAGE

Pluripotent CSF eluted from HPLC (FIG. 3) was treated and processed(under non-reducing conditions) as shown in FIG. 4. Afterelectrophoresis, the gel was sliced into 4 mm sections and proteins fromeach slice were eluted into RPMI 1640 containing 10% FCS. After 18hours, eluted proteins were assayed for pluripotent CSF activity (GM-CSFactivity: shaded area).

FIG. 6: Isoelectrofocusing

HPLC purified lyophilized pluripotent CSF was supplemented with 20%(v/v) glycerol and 2% ampholines (pH 3.5-10) and layered onto theisodense region of an 0-60% gradient of glycerol containing 2%ampholines (pH 3.5-10). After isoelctrofocusing (2,000 V, 24 hours), 5ml fractions were collected and the pH (∘) determined in each fraction.All fractions were subsequently dialyzed and tested for pluripotent CSFactivity (GM-CSF activity: Δ).

We report the purification and biochemical characterization of a humanpluripotent CSF, produced and released constitutively by human cellsespecially tumor cells such as bladder carcinoma cell line 5637 (ATCCHTB-9) and hepatoma cell line SK-HEP-1 (ATCC HTB52). The cell line(5637) was obtained from Jorgen Fogh at Sloan-Kettering institute, 1275York Avenue, New York, N.Y. 10021.

Pluripotent CSF biological properties include differentiation ofprogenitor cells to all major blood types as well as differentiation ofleukemic cells.

Assay for GM-CSF Activity

GM-CSF activity was tested on human bone marrow (BM) cells cultured withserial dilutions of test samples in semi-solid agar. BM from healthyhuman volunteers, who gave informed consent, was diluted 1:5 inphosphate buffered saline (PBS) and separated by density gradientcentrifugation on Ficoll-Hypaque. 10⁵ separated cells were plated in 1ml of 0.3% agar culture medium that included supplemented McCoy's 5Amedium and 10% heat inactivated fetal calf serum (FCS), as described(Broxmeyer, H. E., et al. (1977) Exp. Hematol. 5:87-102). To thismixture serial dilutions of a laboratory standard or test samples(10%;v/v) in RPMI 1640 with 10% FCS were added. Cultures were scored forcolonies (greater than 40 cells/aggregate) and morphology was assessedafter 7 and 14 days of incubation. GM-CSF units were determined fromdose response curves and expressed as U/ml, where 50 U is the CSFconcentration stimulating half-maximal colony number to develop (Nicola,N. A., et al. (1983) J. Biol. Chem. 258:9017-9021).

Assay for Colony Stimulating Factor for BFU-E and CFU-GEMM

The colony assay for human BFU-E and CFU-GEMM was performed aspreviously described (Li Lu, et al. (1983) Blood 61:250-256). Human bonemarrow cells were subjected to a density cut with Ficoll-Hypaque(density 1.077 gm/cm³ ; Pharmacia Fine Chemicals, Piscataway, N.J.) andthe low density cells were suspended in RPMI 1640 containing 10% FCS at2×10⁷ cells/ml and placed for adherence on Falcon tissue cultures dishes(#3003, Becton Dickinson and Co., Cockeysville, Md.) for 1 1/2 hr. at37° C. The nonadherent cells were depleted of T lymphocytes by rosettingwith neuraminidase-treated sheep erythrocytes. Medium conditioned byleukocytes from patients with hemochromatosis in the presence of 1%(v/v) phytohemagglutinin (PHA) (Li Lu, et al. (1983) Supra) as positivecontrol or serial dilutions of test samples were then added at 5% (v/v)to 5×10⁴ of these low density, non-adherent and T lymphocyte depletedbone marrow cells in a 1 ml mixture of Iscove's modified Dulbecco medium(GIBCO Grand Island, N.Y.), 0.8% methylcellulose, 30% FCS, 5×10⁻⁵ M2-mercaptoethanol, 0.2 mM Hemin, and one unit of erythropoietin(Hyclone, or Connaught Labs., Willowdale, Ontario, Canada). The additionof Hemin is necessary to obtain optimal cloning efficiency (Li Lu, etal. (1983) Exp. Hematol. 11:721-729). Dishes were incubated in ahumified atmosphere of 5% CO₂ in air at 37° C. After 14 days ofincubation, colonies were scored and morphology was assessed.

As shown above, a single protein stimulates colony formation byCFU-GEMM, BFU-E, and CFU-GM progenitor cells. This protein we termed"pluripotent CSF". Due to the low numbers of mixed colonies per dishattainable in this assay system, titration of test samples fordetermination of pluripotent CSF activity meets with considerabledifficulties. Therefore, we used the GM-CSF assay units as describedabove to measure the GM-CSF aspect of the pluripotent CSF activity inthose samples that supported growth of BFU-E and CFU-GEM for calculatingthe specific activity throughout the purification procedure.

Differentiation Induction Assay

Titrated samples of purified pluripotent CSF were assayed fordifferentiation induction of WEHI 3B (D+) or HL-60 leukemic cells asdescribed (Metcalf, D. (1980) Int. J. Cancer 25:225-233; Fibach, E., etal., J. Cell Physiol. 113:(1) 152 (1982)).

Rossette Assays for Fc Receptor, OKM1 and Leu M2 antigens

Cell receptors for immunoglobulin Fc were assayed with IgG (CappelLaboratories, West Chester, Pa.) coated sheep erythrocytes as describedelsewhere (Ralph, P., et al. (1983) Blood 62:1169). OKM1 (OrthoDiagnostics Systems Inc., Raritan, N.J.) or Leu M2 (Beckton Dickinson,Mountain View, CA) reactive antigens were detected by incubating 10⁶cells/0.1 ml phosphate buffered saline containing 1.0 ug/ml monoclonalantibody for 20 min at 24° C., washing, incubating 20 min at 24° C. witha a:100 dilution of rabbit anti-rat (Leu M2) or anti-mouse (OKM1) IgGserum (Cappel Laboratories, Cochranville, Pa.), washing and rosettingwith protein A-coated erythrocytes as described previously (Ralph, P. etal., Supra).

Assays for fMLP Receptor

Receptors for chemotactic peptide, formyl-Methionyl-Leucyl-Phenylalanine(fMLP), were assayed as follows; 2×10⁶ cells were incubated with 15 nM ³H-fMLP (New England Nuclear, Boston, Mass.) in a total volume of 0.2 mlin the presence or absence of 10 uM unlabelled fMLP (Sigma Corp, St.Luis, Mo). After three hrs at 4° C. the cell suspensions were rapidlyfiltered onto glass fiber dics (Whatman Inc., Clifton, N.J.), which werethen washed with 30 mls of 4° C. phosphate buffered saline (Harris, P.et al. (1985) Cancer Res. 45:9). Radioactivity on the discs were countedby liquid scintillation spectrophotometry.

Measurement of PMA-Stimulated Hydrogen Peroxide Release

The production of hydrogen peroxide in response to PMA stimulation wasassayed by horse radish peroxidase (HRPO) (Sigma) mediated H₂ O₂dependant oxidation of homovanillic acid (HVA) (Sigma), as described(Harris, et al. Supra (1985). Briefly, cells (1×10⁶) were suspended in 2ml of a solution containing 100 micromolar HVA, 5 U/ml HRPO in theabsence or presence of 30 ng/ml PMA. Following 90 min incubation at 37°C., the incubation was centrifuged and 0.25 ml of 25 mM EDTA, 0.1Mglycine-NaOH, pH 12 was added to the supernates. A 30% stock solution ofhydrogen peroxide (Sigma) was used to prepare H₂ O₂ standards (0.001 to50 nmoles/assay) for the construction of a standard curve. The HVAoxidation product was measured on a Perkins-Elmer Model MPF-44Afluorescence spectrophotometer. Excitation and emission were set at 312nm and 420 nm, respectively.

Prostaglandin Measurements

Cells for prostaglandin production assay were washed three times inphosphate-buffered saline and placed in fresh RPMI 1640 media (withoutFCS) in the presence or absence of 10 micrograms/ml Concanvalin--A(Con-A). Cells were cultured for 24 hrs, centrifuged and the supernatesharvested. Supernates were stored at -20° C. until assayed.

Prostaglandin standards PGE₂, 6-keto-PGF1_(a) and TBX₂ were kindlysupplied by by Dr. J. Pike (Upjohn Company, Kalamazzo, Mich.). Tritiumlabelled compounds were purchased from New England Nuclear (Boston,Mass.). Rabbit antisera to PGE were obtained from the Pasteur Institute(Paris, France). Antibodies to 6-keto PGF1_(a) were raised in thelaboratory (Rashida Karmali). The cross reactivity of these antibodiesfor the non-targeted PGs were to greater than 4% except for the PGE₂antisera which cross reacted 10% with PGE₁ standard. The procedure forextracting the prostaglandins has been described earlier (Karmali, R.A., et al. (1982) Prostagl. Leukotr. Med. 8:565). Briefly, a trace of [³H]-PG was added to aliquots of standard and samples before beingextracted once with petroleum ether. After acidification to pH 3.5, thesamples were extracted twice with diethyl ether, dried under nitrogenand reconstitution in assay buffer. The efficiency of this extractionprocedure to this point was 85-95%. Standard quantities of eachprostaglandin (0-1000 pg) or the extracted sample to be measured wereprepared in 0.1 ml aliquots of assay buffer. Antisera and label wereadded succesively in 0.1 ml aliquots and incubated at 4° C. for 8-12hours. Bound and free [³ H]-PG were separated by 0.5 ml dextran-coatedcharcoal (0.5-1.0% w/v) to estimate the amount of each compound in theunknown sample. The detection limit of this assay has been found to be10 pg. The intra-assay coefficient of variation was 9.0%.

Alkaline and Acid Phosphatase, b-Glucuronidase and N-AcetylGlucuronidase Assays

Cell extracts were prepared in 0.5 ml of PBS 1% NP-40, incubated 5 minat 24° C., then spun for 10 min at 3×10⁴ g. Supernates were collectedand assayed. Extracts were assayed of their alkaline and acidphosphatase, b-glucuronidase and N-acetyl glucuronidase activity usingthe respective Sigma kits. Activities of extracts are expressed aschange in absorbance per unit time per unit sample volume divided by thecell concentration in the culture or in the extract and compared tocontrol activity. Measurements were made in a Beckman ACTA-CVspectrophotometer.

Glycoconjugate assay

Cytokine preparations were assayed in a [³ H]-Glucosamine incorporationassay. Replicate wells were plated with 100 microliters of inducingagent to be tested. Previously washed (3× n PBS) HL-60 or U937 cells(1×10⁷ cells/ml) in RPMI 1640 without FCS were added (50 microliters).After a four hour incubation 20 microliter of 25 uCi/ml [³H]-Glucosamine in 1% BSA (w/v) in PBS was added to the culture andplates were incubated for an additional 16 hours. Cells were harvested(Mini-Mash, Microbiological Associates, Md.) onto glass filter paperwith water wash (×4, 0.1 ml each), followed by 0.4N Perchlorate wash(×4, 0.1 ml) and water (2×, 0.1 ml). Radioactivity on glass discs wasdetermined by liquid scintillation spectrophotometry.

Statistical Analysis

Student's T test to compare means was carried out using the significancelimits of a two tailed test. Preparation of 5637 cell line conditionedmedium (5637 CM)

The human bladder carcinoma cell line 5637 has been reported to producea colony stimulating factor for granulocytes and macrophages(Svet-Moldavsky, G. J., et al. (1980) Exp. Hematol. 8 (Suppl. 7):76).The cell line has been maintained at Sloan-Kettering Institute (NewYork, N.Y.) for several years. It is serially passaged by trypsinizationin the presence of EDTA and grows rapidly to form an adherent monolayerin plastic tissue culture flasks. Routinely, cells are cultured in RPMI1640, supplemented with 2 mM L-glutamine, antibiotics and 10% FCS. Forpurification of pluripotent CSF activity from 5637 conditioned medium(5637 CM), confluent cell cultures were intermittently cultured inmedium containing 0.2% FCS. After 48-72 hours, 5637 CM was harvested,cells and cell debris removed by centrifugation (20 min, 10,000×g), andstored at -20° C. until use.

5637 cells also contain a multitude of subclones which either producep-CSF in better yield and/or have less inhibitor present. Over 120subclones have been isolated. One such subclone 1A6 was found to produceat least twice as much as the parent cell line and possibly 5-10 foldmore resulting in a range of between 2-10 times more p-CSF from the 1A6subclone than from the parent 5637 cell line as as determined by theassay methods outlined. This subclone or the parent cell line 5637 canbe used to isolate p-CSF in good yield. Subclones are isolated bylimiting single dilution techniques to produce a single cell per well inorder to grow up a pure cell line from each well. Best results areobtained if the cells are distributed such that 37% of the wells (oneout of every three) show growth at a certain dilution. There is then agood mathematical chance of obtaining subcloning to obtain outgrowth ofonly one cell from the one of three wells showing growth. Subclone 1A6cell line is on deposit and available at Sloan-Kettering Institute forCancer Research 1275 York Avenue, New York, N.Y. 10021. We refer to useof the 1A6 in a U.S. patent application filed Aug. 23, 1985 Ser. No.768,959 entitled "Production of Pluripotent Granulocytecolony-stimulating Factor" by Lawrence M. Souza to yield sequence dataon the protein p-CSF with subsequent preparation of recombinant p-CSFfrom such a sequenced probe (P11- top P14) as follows:

"(B) Sequencing of Materials Provided by Revised Methods

In order to obtain a sufficient amount of pure material to performsuitably definitive amino acid sequence analysis, cells of a bladdercarcinoma cell line 5637 (subclone 1A6) as produced at Sloan-Ketteringwere obtained from Dr. E. Platzer. Cells were initially culturedIscove's medium (GIBCO, Grand Island, N.Y.) in flasks to confluence.When confluent, the cultures were trypsinized and seeded into rollerbottles (1 1/2 flasks/bottle) each containing 25 ml of preconditionedIscove's medium under 5% CO₂. The cells were grown overnight at 37° C.at 0.3 rpm.

Cytodex-1 beads (Pharmacia, Uppsala, Sweden) were washed and sterilizedusing the following procedures. Eight grams of beads were introducedinto a bottle and 400 ml of PBS was added. Beads were suspended byswirling gently for 3 hours. After allowing the beads to settle, the PBSwas drawn off, the beads were rinsed in PBS and fresh PBS was added. Thebeads were autoclaved for 15 minutes. Prior to use, the beads werewashed in Iscove's medium plus 10% fetal calf serum (FCS) before addingfresh medium plus 10% FCS to obtain treated beads.

After removing all but 30 ml of the medium from each roller bottle, 30ml of fresh medium plus 10% FCS and 40 ml of treated beads were added tothe bottles. The bottles were gassed with 5% CO₂ and all bubbles wereremoved by suction. The bottles were placed in roller racks at 3 rpm for1/2 hour before reducing the speed to 0.3 rpm. After 3 hours, anadditional flask was trypsinized and added to each roller bottlecontaining beads.

At 40% to 50% of confluence the roller bottle cultures were washed with50 ml PBS and rolled for 10 min. before removing the PBS. The cells werecultured for 48 hours in medium A [Iscove's medium containing 0.2% FCS,10⁻⁸ M hydrocortisone, 2 mM glutamine, 100 units/ml penicillin, and 100ug/ml streptomycin]. Next, the culture supernatant was harvested bycentrifugation at 3000 rpm for 15 min., and stored at -70° C. Thecultures were refed with medium A containing 10% FCS and were culturedfor 48 hours. After discarding the medium, the cells were washed withPBS as above and cultured for 48 hours in medium A. The supernatant wasagain harvested and treated as previously described.

Approximately 30 liters of medium conditioned by 1A6 cells wereconcentrated to about 2 liters on a Millipore Pellicon unit equippedwith 2 cassettes having 10,000 M.W. cutoffs at a filtrate rate of about200 ml/min. and at a retentate rate of about 1000 ml/min. Theconcentrate was diafiltered with about 10 liters of 50 mM Tris (pH 7.8)using the same apparatus and some flow rates. The diafilteredconcentrate was loaded at 40 ml/min. onto a 1 liter DE cellulose columnequilibrated in 50 mM Tris (pH 7.8). After loading, the column waswashed at the same rate with 1 liter of 50 mM Tris (pH 7.8) and thenwith 2 liters of 50 mM Tris (pH 7.8) with 50 mM NaCl. The column wasthen sequentially eluted with six 1 liter solutions of 50 mM Tris (pH7.5) containing the following concentrations of NaCl: 75 mM; 100 mM; 125mM; 150 mM; 100 mM; and 300 mM. Fractions (50 ml) were collected, andactive fractions were pooled and concentrated to 65 ml on an Amiconultrafiltration stirred cell unit equipped with a YM5 membrane. Thisconcentrate was loaded onto a 2 liter AcA54 gel filtration columnequilibrated in PBS. The column was run at 80 ml/hr. and 10 ml fractionswere collected. Active fractions were pooled and loaded directly onto aC4 high performance liquid chromatography (HPLC) column.

Samples, ranging in volume from 125 ml to 850 ml and containing 1-8 mgof protein, about 10% of which was hpG-CSF. Samples were loaded onto thecolumn at a flow rate ranging from 1 ml to 4 ml per minute. Afterloading and an initial washing with 0.1M ammonium acetate (pH 6.0-7.0)in 80% 2-propanol at a flow rate of 1/ml/min. One milliliter fractionswere collected and monitored for proteins at 220 nm, 260 nm and 280 nm.

As a result of purification, fractions containing hpG-CSF were clearlyseparated (as fractions 72 and 73 of 80) from other protein-containingfractions. HpG-CSF was isolated (150-300μ) at a purity of about 85±5%and at a yield of about 50%. From this purified material 9 μg was usedin Run #4, an amino acid sequence analysis wherein the protein samplewas applied to a TFA-activated glass fiber disc without polybrene.Sequence analysis was carried out with an AB 470A sequencer according tothe methods of Hewick, et al., J. Biol. Chem., 256, 7990-7997 (1981) andLai, Anal. Chem. Acta. 163, 243-248 (1984). The results of Run #4 appearin Table III.

                  TABLE III                                                       ______________________________________                                         ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                     LeuXX                                                                         ______________________________________                                    

In Run #4, beyond 31 cycles (corresponding to residue 31 in Table III,no further significant sequence information was obtained. In order toobtain a longer unambiguous sequence, in a Run #5, 14 μg of hpG-CSFpurified from conditioned medium were reduced with 10 μl ofmercaptoethanol for one hour at 45° C., then thoroughly dried under avacuum. The protein residue was then redissolved in 5% formic acidbefore being applied to a polybrenized glass fiber disc. Sequenceanalysis was carried out as for Run #4 above. The results of Run #5 aregiven in Table IV.

                                      TABLE IV                                    __________________________________________________________________________     ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                     __________________________________________________________________________

The amino acid sequence given Table IV was sufficiently long (44residues) and unambiguous to construct probes for obtaining hpG-CSF cDNAas described infra." (end quote)

Ammonium Sulfate Precipitation, Ion-exchange-chromatography, Gelfiltration

The first three purification steps ((NH₄)₂ SO₄ -precipitation,Ion-exchange-chromatography on DEAE cellulose, DE 52, Whatman, Clifton,N.J., and gel filtration on AcA 54 Ultrogel, LKB, Inc. Rockland, Md.)were performed as described (Welte, K., et al. (1982) J. Exp. Med.156:454-464) except that AcA 54 was used instead of AcA 44 (see alsoDescriptions of FIG. 1 and 2).

Reverse Phase High-Performance Liquid Chromatography (RP-HPLC)

RP-HPLC was performed with a Waters HPLC system (M 6,000 solventdelivery pumps, model 400 variable wavelength detector, data module anddata processor, Waters, Associates, Milford, Mass.). The separation wasperformed on a uBondapak C18 column (Waters). The buffers used were:Buffer A: 0.9M acetic acid/0.2M pyridine, pH 4.0; buffer B: buffer A in50% 1-propanol (Burdick and Jackson, Lab., Muskegon, Mich.). Acetic acidand pyridine were purchased from Fisher, Scientific Co. The pluripotentCSF containing pool obtained from gel filtration was acidified withacetic acid to pH 4.0 and injected onto the uBondapak C18 column withoutregard to sample volume. The column was washed with buffer A (10 min)and bound proteins were eluted using a steep gradient 0-40% buffer Bwithin the first 20 min and a 40-100% gradient of buffer B in 120 min.The flow rate was adjusted to 1 ml/min and 3 ml fractions werecollected. From each fraction a 0.5 ml aliquot was supplemented with 10%FCS, dialyzed against PBS and tested for pluripotent CSF activity.

Isoelectrofocusing

One ml of the purified pluripotent CSF was supplemented with 20%glycerol (vol/vol) and 2% Ampholines (vol/vol), pH 3.5-10 (LKB Products,Inc.). A 5-60% glycerol density gradient containing 2% Ampholines, pH3.5-10, was layered into a isoelectrofocusing column (LKB 8100). Thepluripotent CSF sample was applied onto the isodense region of thegradient, followed by isoelectrofocusing (2,000 V, 24 hours). Five mlfractions were collected and the pH determined in each fraction. Thefractions were dialyzed against PBS and subsequently tested forpluripotent CSF activity.

Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The discontinuous Tris-glycine system of Laemmli (Laemmli, U.K. (1970)Nature 227:680-685) was used for 1.5 mm slab gels of 15% acrylamide. Thesamples (200 ng lyophilized protein eluted from HPLC) were treated with1% SDS in 0.0625M Tris-HCl, pH 6.8 at 37° C. for 1 hour under bothreducing (5% 2-mercaptoethanol) and non-reducing conditions and thenloaded on the gel. After electrophoresis, gels were stained by theBiorad silver staining method (Biorad Laboratories, Rockville Centre,N.Y.). Apparent molecular weights were determined using proteinstandards ovalbumin (MW 43,000), chymotrypsinogen (MW 25,700),beta-lactoglobulin (MW 18,400), lysozyme (MW 14,300) and cytochrome C(MW 12,300) (Bethesda Research Laboratories, Inc. Gaithersburg, Md.) orfrom Pharmacia Fine Chemicals, Piscataway, N.J. After treatment (seeabove) of lyophilized pluripotent CSF under non-reduced conditions andsubsequent electrophoresis, parallel gels were sliced in 4 mm or 2 mmsections, respectively and proteins from each slice eluted either into0.5 ml RPMI 1640 containing 10% FCS or into phosphate buffered saline(PBS; 20 mM phosphate, 0.15M NaCl). After extensive dialysis, the elutedmaterial was assayed for pluripotent CSF activity.

Protein Assay

The protein content of samples were measured using the Lowry technique(Lowry, O. H., et al. (1951) J. Biol Chem. 193:265-275). For proteinconcentrations lower than 2 microgram/ml, samples were subjected toSDS-PAGE, the protein bands were visualized by the silver stainingtechnique and the protein concentration estimated by comparison with aserial dilution of known amounts of proteins.

The examples shown serve to illustrate the invention without limitingsame.

EXAMPLE I Pluripotent CSF activity in 5637 CM

Confluent layers of 5637 human bladder carcinoma cells, when culturedfor 48-72 hours in the presence of 10% FCS, released into the culturemedium 3,000-10,000 units/ml of GM-CSF activity. Media conditioned inthe presence of 0.2% FCS still contained 10-30% of this activity,whereas in serum free 5637 CM the activity drops below 5% of theactivity obtained in the presence of 10% FCS. Although GM-CSF activityin 5637 CM is readily detectable in soft agar bone marrow cultures, notall batches of unfractionated 5637 CM support in vitro growth of BFU-Eand CFU-GEMM. Four to ten times concentrated 5637 CM support in vitrogrowth of BFU-E and CFU-GEMM. Four to ten times concentrated 5637 CMreduced colony formation by CFU-GM 30-70% indicating the presence ofinhibitor(s) in 5637 CM. Inhibitors were removed after ion-exchangechromatography.

EXAMPLE II Purification of pluripotent CSF

A 20-fold concentration of proteins from the 5637 CM was achieved byprecipitation with ammonium sulfate at 80% saturation. The dialyzedprecipitate was loaded on to a DEAE cellulose (DE 52) column. Boundproteins were eluted with a salt gradient from 0.05-0.3M NaCl in 0.05MTris-HCl, pH 7.8. GM-CSF activity eluted as peak 1 between 0.075M and0.1M NaCl and with a second peak at 0.13M NaCl (FIG. 1). Since only peak1 revealed pluripotent CSF activity, was used only this pool for furtherpurifications. Peak 2 included proteins with only GM-CSF activity. Wecalculated the "fold" purification by measuring the GM-CSF activity ofpluripotent CSF. In the unfractionated CM we could not discriminatebetween GM-CSF activity as part of pluripotent CSF activity and GM-CSFactivity without pluripotent properties. Therefore we considered theGM-CSF activity contained in peak 1 from DE 52 as the starting activity(Table 1).

Since in the subsequent purification schedule GM-CSF, BFU-E and CFU-GEMMactivities copurified in all steps, we named these combined activities"pluripotent CSF" and have used this term thereafter. The proteins ofpeak 1 of DE 52 chromatography (including pluripotent CSF activity) wereconcentrated by dialyzing against 50% (w/v) Polyethylenglycol in PBS andpurified further by ACA 54 Ultrogel gel filtration. The pluripotent CSFactivity eluted in fractions 42-49 as a single peak corresponding to amolecular weight of 32,000 daltons (FIG. 2). This step resulted in a 65%recovery of activities and a 15 fold increase of specific activities(Table 1).

The final step involved chromatography on a reverse phase HPLC column(uBondapak C 18). The majority of proteins did not bind to this columnor eluted at low 1-propanol concentrations (less than 20% 1-propanol;FIG. 3). A minor peak of GM-CSF activity without activity in theCFU-GEMM and BFU-E assays but differentation inducing activity on HL-60leukemic cells was eluted at around 30% 1-propanol. Pluripotent CSFactivity eluted as a single sharp peak at 42% 1-propanol (FIG. 3). Thispurification step resulted in a 600-fold increase of specific activityand a 25% recovery of activity. The protein content of the HPLC fractionwas measured by comparing the density in silver stained SDS-PAGE withprotein standards of known concentrations. Using this measurement, weobtained a specific activity of 1.5×10⁸ U/mg protein and a finalpurification of 9,000-fold, calculated from the first peak of DEAEcellulose chromatography. The overall yield was 6.2%. The purificationtable with the degree of purification of pluripotent CSF as measured byGM-CSF activity, protein content, specific activity and yield isdetailed in Table 1.

The final preparation obtained after HPLC (pluripotent CSF activity peakfraction) was analyzed on a 15% SDS-PAGE gel followed by the sensitivesilver staining technique (FIG. 4). Only one major protein band with amolecular weight of 18,000 was seen under both, reducing (5%2-mercaptoethanol) (FIG. 4) and non-reducing conditions. Since thebuffer system used for HPLC did not allow monitoring the protein elutionpattern by measuring the optical density at 280 nm, we applied proteinsof all active fractions on SDS-PAGE. The density of the stained proteinband at 18,000 MW in the peak and side fractions was proportional to theamount of biological pluripotent CSF activity. After electrophoresisunder non-reducing conditions, a parallel gel was sliced into 4 mmsections and proteins eluted from each slice into RPMI 1640 containing5% FCS. Pluripotent CSF activity was found to be localized in the slicenumber corresponding to 18,000 MW (FIG. 5).

In three additional, independent purification runs, pluripotent CSF hadthe same properties and specific activity as described above. In allthree runs parallel gels were sliced into 2 mm sections, proteins elutedinto PBS and tested for pluripotent CSF activity. Re-electrophoresis ofthe proteins eluted from the slices with pluripotent CSF activity againrevealed one single band in a silver stained gel with a molecular weightof 18,000, identical to that shown in FIG. 4.

However further work using markers from Pharmacia shows the molecularweight of the glycosylated p-CSF to be 19,600. The unglycosylatedrecombinant protein shows a M.W. of 18,800.

The purified CSF was also subjected to isoelectrofocusing analysis usinga 5-60% glycerol gradient in an IEF column and 2% Ampholines, pH 3.5-10.Pluripotent CSF activity was localized in one fraction (5 ml) with anisoelectric point of 5.5 (FIG. 6). The total recovery of pluripotent CSFactivity applied to the column was approximately 20%.

Pluripotent CSF activity did not bind to a Concanavalin A agarose.Treatment with neuraminidase did not abolish the biological activity anddid not change the IEP. However, the isoelectrofocusing under ourconditions did not allow judgment of minor changes of the IEP. Thesefindings suggest that glycosylation might not be a major structuralfeature.

The partial amino acid sequence was determined by Applied MolecularGenetics (Thousand Oaks, Calif.) on an AB 1470A- Beatricemicrosequencer. From the amino terminal end the sequence is Threo, Pro,Leu, Gly, Pro, Ala, Ser, Ser, Leu, Pro. Also see the extended 44 residuesequence above.

EXAMPLE III Biological activity of pluripotent CSF

Progenitor cell stimulation and Effect on Leukemic cells.

1. Progenitor cells:

Fifty unit of GM-CSF activity, enough to support the half maximal growthof CFU-GM, had no clear effect in a CFU-GEMM assay; however, 500 U/ml(GM-CSF activity) of pluripotent CSF clearly supported the growth ofhuman mixed colonies (CFU-GEMM), megakaryocytic colonies, and earlyerythroid colonies (BFU-E) under our experimental conditions (Table IIA& IIB).

Pluripotent-CSF supports the growth of colony forming progenitors of thegranulocyte, mixed granulocyte, macrophage, eosinophil andmegakaryocytic cell types. These results can be seen for example invitro.

We show the results of comparison of 5637-CM and 1A6 -CM in Table IIC atdilutions of 1/10 through 1/1600. The 1/10 dilution of 1A6 shows aninhibitor to be present in the CM. Essentially this table serves as anexample that the 1A6 subclone of 5637 has 8.7 times more p-CSF in U/mlunder growing conditions containing FCS.

When purifying p-CSF the FCS is reduced to 0.2%.

2. Pluripotent CSF also induces the differentiation of leukemic cells.For example, leukemic cell lines HL-60 and WEHI-3B (D+) are induced todifferentiate along the granulocytic and/or macrophage pathway. Thehuman leukemic cell line KG-1 responds to pluripotent CSF by increasedcolony formation in agar and proliferation in liquid suspension culture.

As for mature cells pluripotent CSF induces increased protein content,for example, in macrophages, whereas IL-3 is not reported to be activeon macrophages. (Table III). 50 U/ml and 200 U/ml of GM-CSF activity ofthe pluripotent CSF were needed to induce half-maximal differentiationof the leukemic cell lines WEHI-3B(D+) and HL-60 , respectively. Thesecells were used in a test system (Metcalf Int. J. Cancer (1980) 25:225and Fibach et al. (1982) 113:152) Table IV(A&B) to show the effect ofpluripotent-CSF on leukemic cells. U937 was obtained from Dr. Nilssonand HL-60 from Dr. Gallo as freeze-backs of early passages. HL-60 is amyeloid cell line from an acute promyelocytic leukemia [Gallagher et al.Blood 54:713 (1979)]. U937 is a histiocytic lymphoma cell line(Sundstrom and Nilsson (1976) Int. J. Cancer 17:565).

Differentiation of leukemic cells lines in vitro can be achieved by avariety of nonphysiologic (e.g. DMSO, phorboldiesters) and physiologic(e.g. retinoic acid, vitamin D₃) inducers (Koeffler et al. (1983) Blood62:709). Murine G-CSF is known to be a potent inducer of differentiationof WEHI-3B (D+) murine myelomonocytic leukemia cells, whereasInterleukin 3 lacks this activity (Nicola et al. (1984) Immunol. Today5:76) (See Table V).

Pluripotent-CSF was tested for leukemia differentiating activity (GM-DF)in a clonal assay system described by Metcalf ((1980) Int. J. Cancer25:225; Fibach, E., et al. J. Cell. Physiol. Supra) using murine WEHI-3B(D+) and human HL-60 promyelocytic leukemia cell lines (Platzer et al.(in press) (1985). Quantitation of GM-DF was obtained by incubation ofleukemic cells in agar with serial dilutions of pluripotent CSF.Pluripotent CSF had GM-DF activity on both cell lines. However, HL-60required approximately 2.5-5x higher concentrations of Pluripotent CSFto achieve 50% differentiated, spreading colonies versusundifferentiated tight blast cell colonies, than did WEHI-3B (D+)(Platzer et al. 1985, Supra).

Morphological and cytochemical analysis of HL-60 colonies were performedusing alpha-naphthylacetate esterase (ANAE) and luxol fast blue (LFB)stains, as markers of the monocyte, macrophage and eosinophilgranulocyte lineage respectively (Platzer, E., et al. J. Immunol. inpress). In the presence of pluripotent CSF there is observed an increasein the number of colonies containing polymorphonuclear cells (byhematoxylin stain), LFB⁺ cells and in intensity of ANAE stain. Thereforepluripotent CSF induces differentiation along the macrophage as well asgranulocyte pathway. The human leukemia cell line KG1 (courtesy Dr. H.P. Koeffler) responded to Pluripotent CSF in a dose dependent mannerwith increased colony formation in agar and increased ³ H-thymidineincorporation after 24-48 hrs. in suspension culture. This mightindicate that the GM-DF activity of Pluripotent CSF reflects thedifferentiating capacity of a given leukemic cell lines rather than anintrinsic property of the factor.

CM from SK-HEP and cell line 5637 containing pluripotent CSF (free ofInterferon) has also shown acquisition of immunoglobulin Fc receptor,growth inhibition, increased expression of monocyte related surfaceantigens and an increase in lysosomal enzyme content as well as (todistinguish P-CSF from Interferon-gamma) increased receptors forchemotactic peptide, increased hydrogen peroxide release in response tophorbol myristic acetate (PMA) stimulation and the release ofprostaglandins (PGE and 6-keto PGF_(1A)) as features of differentiationof human promyelocytic leukemia cell line HL-60 and monoblastic leukemiacell line U937. These broad range differentation factors are thusdifferent from Interferon and conventional colony stimulating activity(CSA) (Harris et al. submitted). Highly purified pluripotent CSFincreased the receptors for chemotactic peptide and increasedglycoconjugate synthesis as a feature of differentiation in both thehuman promyelocytic leukemia cell line HL-60 and monoblastic leukemiccell line U937.

3. Pluripoietin CSF shows species crossreactivity on normal murine bonemarrow and leukemic cells:

Normal mouse bone marrow cells cultured in agar for 7 days in thepresence of saturating concentrations of Pluripoietin formedapproximately 10% of the colonies supported by WEHI-3B conditioned mediaas standard source of CSF('s). All colonies formed in the presence ofPluripotent CSF were of similar morphology, not staining foralpha-naphthyl-acetate esterase or Kaplow's myeloperoxidase; thissuggests that a subpopulation of murine colony forming progenitors isresponsive to Pluripotent CSF. Weak cross species activity was found oncontinuous murine mast cell lines, established as described from murinelong-term bone marrow cultures (Tertian et al. (1980) J. Immunol.127:788). 5,000 cells/well of a mast cell growth factor (MCGF) dependentmurine mast cell line were incubated for 24 hrs. at 37° C. in 96 wellplates with serial dilutions of growth factors, and then assayed for ³H-thymidine uptake as described (Yung et al. (1981) J. Immunol.127:794). Results demonstrate little more than 10% murine MCGF activityof Pluripotent CSF as compared to ConA-LBRM CM, which was used as astandard preparation of murine MCGF. The murine Interleukin 3 dependentcell line FDC-P2 (courtesy Dr. M. Dexter) did not respond with increased³ H-thymidine uptake to concentrations of Pluripoietin as high as 2,000U/ml.

We herein describe the purification of a pluripotent CSF, which isconstitutively produced by the human bladder carcinoma cell line 5637,its 1A6 subclone or SK-HeP-1. This protein is capable of stimulating thein vitro growth of mixed colony progenitor cells (CFU-GEMM), earlyerythroid progenitor cells (BFU-E), megakaryoCytic (CFU-Mega),granulocyte-macrophoage progenitors (CFU-GM) and in addition inducesdifferentiation of both the murine myelomonocytic (WEHI- 3B(D+)) and thehuman promyelocytic (HL-60 ) leukemic cell lines (E. Platzer, K. Welte,J. Gabrilove, Li Lu, M. A. S. Moore, manuscript in preparation). Thepurified pluripotent CSF had a specific activity in the GM-CSF assay of1.5×10⁸ U/mg protein. To our knowledge this is the highest specificactivity for a human pluripotent CSF reported to date. Pluripotent CSFhas a molecular weight of 32,000 by gel filtration and 18,000 bySDS-PAGE under both, reduced and non-reduced conditions and anisoelectric point of 5.5. Pluripotent CSF activities could be elutedfrom gel slices representing the same molecular weight range as thestained protein band.

The purified protein shown in SDS-PAGE is consistent with pluripotentCSF because: 1) The profile of protein elution visualized in SDS-PAGEand elution of pluripotent CSF activity (FIG. 3) from reverse phase HPLCcolumns is equivalent in the major fraction and side fractions; 2)additional chromatography of the purified protein on diphenyl or octylreverse phase HPLC columns using acetonitrile or ethanol as organicsolvents for elution did not lead to a separation of protein andpluripotent CSF activity; 3) identical localization of protein band andpluripotent CSF activity in a preparative SDS-PAGE; 4) high specificGM-CSF activity (1.5×10⁸ U/mg protein). Since purified pluripotent CSFis apparently homogeneous, amino acid sequence analysis of the purifiedprotein has been initiated and is partially determined.

Based on the molecular weight of pluripotent CSF as 18,000 it could becalculated that 1 U of pluripotent CSF was equivalent to 6.7 pg proteinor 3.7×10⁻¹⁶ moles. A pluripotent CSF concentration of 50 U/ml or1.85×10⁻¹¹ M was required for half maximal colony formation for CFU-GMactivity in normal human bone marrow cells.

A ten-fold increase in the amount of pluripotent CSF (500 U/ml GM-CSFactivity) was required for clear detection of human CFU-GEMM anderythroid BFU-E activities (Table II); a 1-2 or 1-2.5 fold increase inpluripotent CSF (e.g. 50-200 U GM-CSF) was needed to induce thedifferentiation of either WEHI-3 B (D+) or HL-60 leukemic cells,respectively. These data suggest that the particular action(s) ofpluripotent CSF are determined by its concentration as first suggestedby Burgess and Metcalf (Blood, Supra) in the murine system. The factthat human pluripotent CSF is able to induce differentiation of leukemiccell lines makes it a protein with unique properties, since for themurine multi CSF (Interleukin 3) no differentiation activity on leukemiccells has been reported (Ihle, J. N., et al. (1982) J. Immunol.129:2431-2436; Nicola, et al. (1984) Immunol. Today 5:76, Watson, et al.(1983) Immunol. Today 5:76, and Fung et al. (1984) Nature 307:233).(Table V compares the two entities). The murine IL-3 dependent cell lineFDC-P2 (Dr. M. Dexter) did not respond with increased ³ H-thymidineuptake to Pluripotent-CSF as high as 2,000 U/ml.

Several human CSFs (GM-CSF, G-CSF, eosinophilic CSF, erythroidpotentiating activity) have molecular weights between 30,000 and 40,000on gel filtration (Nicola, N. A., et al. (1979) Blood 54:614-627; Golde,D. W., et al. (1980) Proc. Nat'l. Acad. Sci. USA 77:593-596; Lusis, A.J., et al. 1981) Blood 57:13-21; Abboud, C. N., et al. (1981) Blood58:1148-1154; Okabe, T., et al. (1982) J. Cell. Phys. 110:43-49) whichis similar to the native molecular weight of the pluripotent CSFdescribed here. However, only partially purified erythroid-potentiatingactivity has been reported to have activity in a CFU-GEMM assay (Fauser,A. A., et al. (1981) Stem Cells 1:73-80).

Constitutive production of pluripotent CSF by the bladder carcinoma cellline 5637 and its 1A6 subclone or other 5637 subclones suggests thatthese are valuable source for large scale production and for isolationand cloning of the gene which codes for pluripotent CSF. Theavailability of purified human pluripotent CSF has important and farreaching implications in the management of clinical diseases involvinghematopoietic derangement or failure, either alone or in combinationwith other lymphokines or chemotherapy. Such disorders include leukemiaand white cell disorders in general. It is useful in transplantation,whether allogeneic or autologous, to augment growth of bone marrowprogenitor cells. It can be used in induced forms of bone marrow aplasiaor myelosuppresion, in radiation therapy or chemotherapy-induced bonemarrow depletion, wound healing, burn patients, and in bacterialinflammation. Here the action of pluripotent-CSF may possibly be due toenhancement of chemotactic peptide receptors or by functioning as achemo-attractant. It is also found in saliva so may prevent tooth decayand oral infection.

p-CSF may be used alone or together with recombinant material or inconjunction with erythropoietin for treatment in hematopoieticdisorders.

                  TABLE I                                                         ______________________________________                                        Purification of human pluripotent CSF                                                          Total     Specific                                                                              Purifi-                                                     activity.sup.a                                                                          activity                                                                              cation                                                                              Yield                                Fraction                                                                              Protein  (U × 10.sup.-6)                                                                   (U/mg)  (fold)                                                                              (%)                                  ______________________________________                                        5637 CM 2      g     12        6 × 10.sup.3                                                                  --    100                                DEAE-   300    mg    5       1.7 × 10.sup.4                                                                  1.sup.b                                                                             42                                 cellulose                                                                     AcA 54  13     mg    3.1     2.4 × 10.sup.5                                                                  14    26                                 Ultrogel                                                                      RP-HPLC 5      μg 0.74    1.5 × 10.sup.8                                                                  9,000 6.2                                ______________________________________                                         .sup.a GM-CSF activity of pluripotent CSF; U = Units                          .sup.b estimate of fold purification based on starting activity of peak 1     of DEAE cellulose chromatography                                         

                  TABLE IIA                                                       ______________________________________                                        Comparison of CFU-GEMM and BFU-E activities                                   of pluripotent CSF (500 U/ml GM-CSF activity)                                        CFU-GEMM.sup.a BFU-E.sup.a                                                    (Colonies ± 1 SEM)                                                                        (Colonies ± 1 SEM)                                   Experi-                                                                       ment #:  1       2        3     1     2     3                                 ______________________________________                                        Medium   0.3 ±                                                                              0        0     42 ± 6                                                                           17 ± 3                                                                           17 ±                                    0.3                                2                                 PHA-LCM.sup.b                                                                          7 ±  3 ± 0 3.3 ±                                                                            67 ± 1                                                                           65 ± 3                                                                           34 ±                                    1                0.3               3                                 Pluri-   7.7 ±                                                                              4 ± 0.8                                                                             2.3 ±                                                                            85 ± 6                                                                           31 ± 1                                                                           28 ±                           potent CSF                                                                             2.1              0.9               2                                 ______________________________________                                         .sup.a Target cells were 5 × 10.sup.4 /ml low density, nonadherent      and T cell depleted normal human bone marrow cells. Experiment 3 was done     in the absence of Hemin.                                                      .sup.b Medium conditioned by leukocytes from patients with hemochromatosi     in the presence of 1% PHA. (positive control)                            

                  TABLE IIB                                                       ______________________________________                                        Activity of Pluripoietin on pre-CFU                                                  Exp. 1  Exp. 2    Exp. 3                                                        7 days in 7 days in 5 days in                                                                             9 days in                                Pluripoietin                                                                           supension suspension                                                                              suspension                                                                            suspension                               concentration                                                                          culture   culture   culture culture                                  ______________________________________                                        1000 U/ml                                                                              416 ± 18                                                                             20 ± 4 32 ± 5                                                                             80 ± 8                                500 U/ml 367 ± 57                                                                             39 ± 4 n.t.    n.t.                                     100 U/ml n.t.      29 ± 6 73 ± 4                                                                             30 ± 5                                 10 U/ml n.t.      12 ± 3 52 ± 3                                                                             34 ± 4                                Control  200 ± 16                                                                              8 ± 2 26 ± 5                                                                             20 ± 4                                medium                                                                        ______________________________________                                    

                                      TABLE IIC                                   __________________________________________________________________________    COMPARISON OF ACTIVITY OF 5637 AND 1A6 IN GM CFU ASSAY                                  Dilution                                                            Cells     1/10 1/100                                                                             1/200                                                                              1/400                                                                              1/800                                                                             1/16000                                                                            U/ml                                    __________________________________________________________________________    5637-CM                                                                            Colonies                                                                           190 ± 17                                                                        75 ± 1                                                                         18 ± 6                                                                           0    0   0   2750 ±                                    %    100% 39% 9%                 350                                               (max)                                                                              of max.                                                        1A6  Colonies                                                                            0 ± 0                                                                          93 ± 7                                                                         130 ± 11                                                                        124 ± 0                                                                         64 ± 0                                                                         30 ± 6                                                                          24,000 ±                                  %     0%  49  69   65   34  16    3,000                                  __________________________________________________________________________

Legend Table II

Normal human bone marrow cells were separated by Ficoll, adherence toplastic and depletion of T cells by rosetting with neuraminidase treatedsheep red blood cells, as described (Platzer, E., et al. J. Immunol./inpress). Quadruplicate cultures of 25,000 cells in 100 microliters/wellwere incubated in 96 well flat bottom tissue culture plates in Iscove'smodified Dulbecco's medium supplemented with 30% fetal bovine serum(FBS), 5×10⁻⁵ M 2-mercapto-ethanol and serial dilutions of purifiedPluripoietin or control medium for 5, 7 or 9 days at 37° C. in 5% CO₂ inair. Contents of each well were then resuspended and incorporated into 1ml agar system in supplemented McCoy's with saturating concentrations(10% v/v) of 5637 CM, as described (Platzer, E., et al. 1985 Supra,Welte, K., et al. (1985) Proc. Nat'l. Acad. Sci. U.S.A. in press).Colonies were scored after 7 days of incubation at 37° C. in ahumidified atmosphere of 5% CO₂ in air. Results are expressed as meancolony number per well ±1 standard deviation. CFU input on day 0 were79±5 (exp. 1), 26±1 (exp. 2) and 22±3 (exp. 3) per well. Bone marrowcells from the donor for experiment 1 grew high numbers of CFU-GM in twounrelated experiments; no pathophysiological situation was recognized.

                  TABLE III                                                       ______________________________________                                        Influence of Pluripoietin on protein content                                  in cultures of human macrophages                                                       Adherent cell protein                                                                        Adherent cell protein                                          in response to in response to                                        Time in  control medium Pluripoietin                                          culture  μg/coverslip                                                                              μg/coverslip                                       ______________________________________                                        Day 1-2  10.0 ± 2.0  28.6 ± 7.7                                         Day 1-3  20.4 ± 1.6  26.8 ± 2.5                                         Day 1-4  28.4 ± 1.6  41.2 ± 1.9                                         Day 4-5  28.8 ± 1.6  28.1 ± 3.6                                         Day 4-6  43.1 ± 4.7  28.1 ± 3.6                                         Day 4-7  38.2 ± 6.1  44.8 ± 0.7                                         ______________________________________                                    

Legend Table III

Normal human monocytes/macrophages were isolated from peripheral bloodmononuclear cells by adherence to glass surfaces ²⁵. Two×10⁶ cells wereplated per 13 mm diameter coverslips in 0.1 ml of supplemented RPMI 1640containing 25% fresh frozen human serum. After 2 hrs. at 37° C.,nonadherent cells were removed by rinsing, and coverslips transferred to24 well tissue culture medium containing 500 U/ml of purifiedPluripoietin or control medium. Protein content was determined 1 to 3days thereafter by rinsing coverslips free of culture medium,solubilizing adherent cell protein in 0.5N NaOH and measuring proteinconcentration according to the method of Lowry.* Results are expressedas mean ±1 standard deviation, from triplicate cultures.

                  TABLE IVA                                                       ______________________________________                                        Leukemia differentiating (GM-DF) activity of purified Pluripoietin            GM-CSF activity                                                                              GM-DF activity                                                                             GM-DF activity                                    Puri-                                                                              Specific          WEHI-3B (D+)                                                                             HL-60                                       fica-                                                                              activity U/             Ratio        Ratio                               tion mg protein                                                                              U/ml    U/ml  DF/CSF U/ml  DF/CSF                              ______________________________________                                        I     1.5 × 10.sup.8                                                                    84,000 246,000                                                                             2.9    54,000                                                                              0.6                                 II   1.25 × 10.sup.8                                                                   201,000 502,000                                                                             2.5    80,000                                                                              0.4                                 ______________________________________                                    

                  TABLE IVB                                                       ______________________________________                                        Glycoconjugate Synthesis                                                                         HL-60   U937                                               Inducer              CPM/5 × 10.sup.5 cells                             ______________________________________                                        media                         465     210                                     gIFN        500    U/ml      1029a   1500a                                                100    U/ml       800a    537a                                                50     U/ml       410     258                                     LK 50%                        427     910a                                    (500 U/ml gIFN)                                                               5637 CM (GM-CSA)                                                                          2      kU/ml     1828a   1200a                                                1      kU/ml      980a    780a                                                500    U/ml       670a    490a                                    fluripoetin.sup.b 1KU                                                                              4235a     2400a                                          pp aCSF.sup.c 1KU     430       306                                           SK-Hep CM       50%      1439a      604a                                      GCT-CM         100%       420       200                                       PMA         3.0    ng/ml      490a    250                                                 50.0   ng/ml     2000a   1700a                                    aIFN        5000   U/ml       420     240                                     IL-2        100    U/ml       425     230                                     ______________________________________                                    

Glycoconjugate synthesis was measured as follows, cells (5×10⁵) wereincubated with inducers for 4 hrs then glucosamine incorporation wasevaluated after an additional 16 hr.

Results are mean values from three or more experiments

a, Significantly different from control, p less than 0.05 by Students Ttest.

b, Human P-CSF, units assigned by CFU_(c) activity. c, partiallypurified aCSF-like activity, units assigned by CFU_(c) activity.

Legend Table IVA

For determination of specific activity, protein concentration ofpurified Pluripoietin was estimated by comparision with serial dilutionsof known amounts of protein in SDS-PAGE, visualized by silver stain. Dueto the low frequency of CFUGEMM in normal human bone marrow cells, thebiological activity of Pluripoietin had to be measure using the GM-CSFassay. We compared the ability of serial dilutions of Pluripoietin and apreviously determined laboratory standard of 5637 CM to supportGM-colony formation in 1 ml semi-solid agar cultures containing 10⁵ lowdensity, normal human bone marrow cells. Fifty units of GM-CSF activitywere arbitrarily defined as inducing 50% of maximal colony growth on day7 of culture. Concentrations of 500 U/ml of Pluripoietin were sufficientto stimulate colony growth from CFU-GEMM and BFU-E comparable to thatsupported by optimal amounts of phytohemagglutinin-activated lymphocyteconditioned media. Two independent purifications (I and II) resulted invery similar specific activity. Due to different amounts of startingmaterial, the final concentration of biological activity of startingmaterial, the final concentration of biological activity differs betweenI and II, but is useful for comparision of GM-CSF and leukemiadifferentiating 3×10² /ml WEHI-3B(D+) or10³ /ml HL-60 leukemic cells in0.3% agar in McCoy's medium containing 12.5% FBS with serial dilutionsof Pluripoietin. Cultures were scored on day 7 (WEHI-3B) and day 14(HL-60) for induction of disperse, differentiated colonies vs. tight,blast cell colonies (Metcalf, et al. (1980) Int. J. Cancer 25:225 andFibach, et al. (1982) J. Cell. Physiol. 113:152). Fifty units of GM-DFactivity were defined as inducing 50% differentiated colonies.

                  TABLE V                                                         ______________________________________                                        Biological activities of purified human Pluripoietin                          and murine Interleukin 3.                                                     Activity        Pluripoietin.sup.a)                                                                       Interleukin 3.sup.b)                              ______________________________________                                        Clonal growth of                                                              hemopoietic progenitors:                                                      CFU-GEMM        +           +                                                 BFU-E           +           +                                                 CFU-G, M, GM    +           +                                                 CFU-EOS         +           +                                                 CFU-MEG         n.t.        +                                                 pre-CFU-c (ΔGPA)                                                                        +           n.t.                                              stem cell multi-                                                                              §      +                                                 plication (CFU-s)                                                             Species crossreactivity.sup.c)                                                                +           -                                                 Leukemia differentiating                                                      activity (GM-DF) on:                                                          WEHI-3B (D+)    +           -                                                 HL60            +           -                                                 .sup.3 H-TdR uptake in cell lines:                                            KG1             +           -                                                 FDC-P2          -           +                                                 Murine mast cell lines                                                                        +           +                                                 (MCGF activity)                                                               Histamine production                                                                          n.t.        +                                                 Protein synthesis of                                                                          +           n.t.                                              mature macrophages                                                            Induction of 20αSDH                                                                     §      +                                                 Growth of:                                                                    natural cytotoxic cells                                                                       §      +                                                 pre-B cell clones                                                                             n.t.        +                                                 ______________________________________                                         .sup.a) Pluripoietin was tested on human target cells, if not noted           otherwise.                                                                    .sup.b) Interleukin 3 activity on murine target cells, if not noted           otherwise. Data derived from literature, except GMDF and activity on KG1.     .sup.c) Activity on bone marrow derived colony formation in agar cultures     § No human test system available                                         n.t. Not tested                                                          

What is claimed:
 1. A preparation of isolated and purified humanpluripotent colony stimulating factor comprising the followingcharacteristics:a) a molecular weight of about 19,600 daltons underreducing and non-reducing conditions as determined by SDS-PAGE; b) amolecular weight of about 32,000 daltons as determined by gelfiltration; c) an isoelectric point of about 5.5; d) the ability toinduce differentiation of the leukemia cell line WEHI-3B (D+); e) theamino-terminal amino acid sequenceThr-Pro-Leu-Gly-Pro-Ala-Ser-Ser-Leu-Pro-Gln-Ser-Phe-Leu-Leu-Lys-Cys-Leu-Glu-Gln-Val-Arg-Lys-Ile-Gln-Gly-Asp-Gly-Ala-Ala-Leu-Gln-;and f) the ability to stimulate growth of both human and mouse bonemarrow cells.